The present study aimed to investigate the karyotype and molecular sex differences in Brycinus nurse. Analysis of 430 metaphases spreads from 12 specimens revealed that the diploid chromosome number is 44 chromosomes and the proposed karyotype formula is n = 3 M + 3 SM + 16 A, FN = 56. No morphologically differentiated heteromorphic sex chromosomes were observed. On the other hand, 29 primers of three molecular markers (RAPD, SCoT and ISSR) were tested to differentiate between males and females of B. nurse. From all tested primers only two primers (OPI-18 and SCoT-18) successfully generated female specific bands in the bulked DNA samples which further confirmed using individual DNA samples. According to molecular analysis result in this study it seems that females of B. nurse are the heterogametic gender (ZW) and males of B. nurse are the homogametic gender (ZZ). In the future, SCoT marker can be used as suitable and powerful marker in genetic analysis studies in fish.